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ORIGINAL ARTICLE
To cite this article: Gerdes VEA, Kremer Hovinga HA, Ten Cate H, MacGillavry MR, Leijte A, Reitsma PH, Brandjes DPM, Buller HR on behalf of the
Amsterdam Vascular Medicine Group. Homocysteine and markers of coagulation and endothelial cell activation. J Thromb Haemost 2004; 2: 44551.
Introduction
Summary. Objective: In vitro studies suggest an inuence of
hyperhomocysteinemia on the coagulation system, but the
inuence of mild hyperhomocysteinemia in vivo is unclear.
Methods and results: We studied the relation between homocysteine and markers of coagulation activation and endothelial
cell activation in 279 patients with established atherosclerotic
disease. In addition, we performed an investigator-blinded
placebo-controlled cross-over study to investigate the inuence
of acute hyperhomocysteinemia by oral methionine load on
these markers in 20 healthy volunteers. In the atherosclerotic
patients prothrombin fragment F1+2 and soluble thrombomodulin (sTM) were associated with homocysteine in univariate analyses (P 0.003 and P 0.001, respectively), but not in
multivariate analyses. Age, creatinine and MTHFR C677T
polymorphism were major determinants of homocysteine
concentration. MTHFR C677T polymorphism status was not
associated with F1+2 and sTM. Median homocysteine
concentrations increased in the healthy volunteers after methione load. However, after methionine load or after placebo,
we did not observe different plasma concentrations of
F1+2 (0.9 nmol L)1 vs. 0.9 nmol L)1, P 0.39), d-dimer
(153 lg L)1 vs. 151 lg L)1, P 0.63) and von Willebrand
factor (103% vs. 107%, P 1.00). Conclusion: These results
provide evidence against a major effect of mild hyperhomocysteinemia on activation of the coagulation system and endothelial cell activation in vivo.
Keywords: atherosclerosis, coagulation, endothelium, homocysteine, methionine load.
446 V. E. A. Gerdes et al
The B-mode ultrasound intima-media thickness (IMT) measurement procedures have been described elsewhere [30].
B-mode ultrasound scans were performed by one sonographer.
An ATL Ultramark IV (Advanced Technology Laboratories,
Bothell, WA, USA) with a High Resolution Linear Array
7.5-MHz transducer was used. Subjects were scanned in the
reclined position. Three right carotid, three left carotid, two
right femoral and two left femoral artery wall segments were
scanned. Images of each arterial wall segment were stored on
S-VHS video tape. IMT of the posterior wall segments was
measured off-line [31]. The average of the 10 segments was used
for analysis. The sonographer and image analyst were blinded
to the clinical status of the subjects.
Statistical analysis
A total of 307 consecutive patients with manifest atherosclerotic disease were included. Homocysteine levels were determined in 279 patients from whom fasting plasma samples were
available. Of these 279 patients, 95 had a recent ischemic
stroke, 86 had a recent myocardial infarction and 98 had
peripheral arterial disease. The mean age was 62.9 years.
Median homocysteine concentration was 15.1 lmol L)1. We
divided the group in quartiles according to homocysteine
n
Age (years)
Male (%)
Diabetes (%)
Smoking (%)
Hypertension (%)
Creatinine (lmol L)1)
Total cholesterol (mmol L)1)
LDL (mmol L)1)
HDL (mmol L)1)
Triglycerides (mmol L)1)
F1+2 (nmol L)1)
sTM (ng mL)1)
IMT (mm)
All patients
II
III
IV
279
63
63
11
85
41
101.7
88.4113.2
6.1
5.46.9
4.0
3.54.7
1.11
0.961.35
1.7
1.22.3
1.33
1.041.73
8.9
7.011.5
0.94
0.771.11
68
58
54
15
78
38
96.0
85.9106.1
6.0
5.46.8
3.9
3.44.7
1.11
0.951.32
1.7
1.22.3
1.09
0.941.41
7.8
6.510.2
0.89
0.751.08
71
62
61
4
87
39
98.1
87.5112.3
6.3
5.77.0
4.0
3.34.6
1.12
0.961.43
1.8
1.22.4
1.34
1.101.67
8.5
6.611.5
0.92
0.721.09
69
64
77
19
86
46
106.0
92.8121.6
6.1
5.46.9
4.1
3.55.0
1.10
0.941.29
1.7
1.42.4
1.25
1.011.88
9.3
7.712.3
1.01
0.781.18
71
68
62
6
87
46
106.1
89.3125.5
6.2
5.46.8
4.1
3.54.7
1.16
0.981.32
1.6
1.32.3
1.47*
1.281.89
9.6*
7.212.8
0.97
0.811.14
sTM, Soluble thrombomodulin; IMT, intima-media thickness. For laboratory values and IMT medians and interquartile ranges are depicted. I:
Homocysteine < 11.8 lmol L)1; II: 11.815.1 lmol L)1; III: 15.118.8 lmol L)1; IV: 18.8 lmol L)1. IMT measurement was performed in 226
patients. P < 0.001; *P < 0.01 (KruskalWallis test).
2004 International Society on Thrombosis and Haemostasis
448 V. E. A. Gerdes et al
between homocysteine and F1+2 was not statistically signicant any more, whereas age and type of arterial disease were the
main determinants of F1+2 in these patients. In the linear
regression analysis with sTM as dependent variable, univariate
analysis selected age, diabetes, homocysteine (B 4.26, 95%
condence interval 1.77, 6.76, P 0.001), lipoprotein(a),
F1+2, creatinine, IMT and type of arterial disease as variables
associated with sTM. In the multivariate model, homocysteine
was excluded and age, F1+2, lipoprotein(a) and peripheral
arterial disease had an independent relationship with sTM.
MTHFR polymorphism in patients with atherosclerotic disease
Hcy (lmol L )
F1+2 (nmol L)1)
sTM (ng mL)1)
IMT (mm)
CC (n 145)
TC (n 107)
TT (n 24)
14.3
11.717.5
1.33
1.011.71
9.4
7.112.2
0.96
0.741.14
16.1
11.719.1
1.32
1.061.69
8.2
6.911.1
0.91
0.791.09
19.8*
14.627.9
1.53
1.031.89
9.6
6.413.6
0.97
0.861.13
Table 3 Homocysteine and markers of coagulation in healthy volunteers, before and after methionine load or placebo
Placebo
VWF
(%)
(lg L)1)
Methionine
t0
t4
t0
t4
8.9
8.210.3
1.00
0.701.16
1.88
1.443.26
146
140259
103
80122
9.2
8.211.0
0.86
0.621.11
1.59
1.252.86
153
123254
103
77121
9.3
7.910.7
1.00
0.691.55
2.36
1.173.07
158
102237
101
75122
30.1
26.737.8
0.86
0.681.27
1.85
0.953.23
151
107235
107
74124
0.000
0.39
0.91
0.63
1.00
Hcy, Homocysteine; TAT, thrombinantithrombin complexes; VWF, von Willebrand factor. Medians and interquartile ranges are depicted.
P-value for comparison of difference (value at t 4 ) value at t 0) after methionine and placebo.
2004 International Society on Thrombosis and Haemostasis
450 V. E. A. Gerdes et al
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